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382 pCMVK-IRES-TagGFP
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Cloning of pCMVK-IRES-TagGFP<br> Purpose: to clone pCMVK-IRES-TagGFP by PCR<br> Date: 2011-9-14---9-17 Notebook:Lu yingying<br> Page:003896---003903 Clone ID:382 Experimenters: lu yingying<br> == Results<br> == [[Image:382 TagGFP PCR 9 15 2011.JPG]]<br>All bands show PCR product of IRES-TagGFP,<br> template:TagGFP-F3R3-PET28b(2)<br> Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG<br> [[Image:382TagGFP+PCMVK9-15-2011.JPG|293x268px|382TagGFP+PCMVK9-15-2011.JPG]]<br> Digest result:Xho1+BglII<br> band1,2,3 show PCMVK vector, band 4,5,6 show IRES-TagGFP Gene Synthesis Colony Screening <br> Result: no GFP fluorescence when transfected into cells.<br> <br> Restart:<br> Date: 2011-9-23--- Notebook:Lu yingying Page:003911--- Clone ID:382 Experimenters: lu yingying<br>Results<br> [[Image:382TagGFP-1stPCR -9-23-2011.JPG|238x130px|382TagGFP-1stPCR -9-23-2011.JPG]] bands show 1st round PCR product <br> template with gene synthesis method:TagGFP oligo mixture of TagGFP1 to TagGFP 20 <br> Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG<br> 25 cycles<br> [[Image:382TagGFP 2nd PCR product-9-23-2011.JPG|255x155px|382TagGFP 2nd PCR product-9-23-2011.JPG]]<br> bands show 2nd round PCR product<br> 1st round PCR product as template, Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG 20 cycles<br> Gene Synthesis Colony Screening Result: no GFP fluorescence when transfected into cells. <br> Restart: Date: 2011-9-26--- Notebook:Lu yingying Page:00--- Clone ID:382 Experimenters: lu yingying<br> Result:<br> [[Image:382TagGFP-PCR -9-26-2011.JPG|291x110px|382TagGFP-PCR -9-26-2011.JPG]]<br> band1,2,3 template:TagGFP-F3R3-PET28b(2)<br> Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG 25 cycles<br> band 4,5,6 template with gene synthesis method:TagGFP oligo mixture of TagGFP1 to TagGFP 20 Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG 25 cycles<br> [[Image:382TagGFP-digest-9-26-2011.JPG|268x182px|382TagGFP-digest-9-26-2011.JPG]]<br> digest :Xho1+BglII<br> [[Image:382-PCMVK digest-9-26-2011.JPG]]<br> digest :Xho1+BglII<br> <br> restart<br> Date: 2011-9-27--- Notebook:Lu yingying Page:00--- Clone ID:382 Experimenters: lu yingying Result<br> :[[Image:382TagGFP PCR product-9-27-2011.JPG|393x126px|382TagGFP PCR product-9-27-2011.JPG]]<br> band1,2,3,4 template:TagGFP-F3R3-PET28b(4) Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG 25 cycles<br> [[Image:344-382-9-27-2011.JPG|336x178px|344-382-9-27-2011.JPG]]<br> band 6,7 show TagGFP digest result<br> band 8 shows PCMVK digest result<br> Xho1+BglII<br> Result: only two clones in plate<br> <br> restart Date: 2011-9-28--- Notebook:Lu yingying Page:00--- Clone ID:382 Experimenters: lu yingying Result<br> [[Image:382TagGFP-PCR product9-28-2011.JPG|247x153px|382TagGFP-PCR product9-28-2011.JPG]]<br> band1,2,3,4 template:TagGFP-F3R3-PET28b(4) Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG<br>Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG 25 cycles<br> [[Image:382TagGFP+PCMVK-digest9-28-2011.JPG|211x198px|382TagGFP+PCMVK-digest9-28-2011.JPG]]<br> bands show digest result,digest :Xho1+BglII<br> [[Image:382TagGFP-PCMVK digest detecting 9-30-2011.JPG|286x221px|382TagGFP-PCMVK digest detecting 9-30-2011.JPG]]<br> TagGFP-PCMVK digest result:Xho1+BglII<br> upper bands show PCMVK vector lower bands show TagGFP == Transfection Result:<br> == clone 2 ,3 , 4 have green fluorescence --[[User:Luyingying|Luyingying]] 14:31, 1 October 2011 (EST) [[Category:Cloning]]
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